Verheul Henk M W, Lolkema Martijn P J, Qian David Z, Hilkes Yvonne H A, Liapi Eleni, Akkerman Jan-Willem N, Pili Roberto, Voest Emile E
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.
Clin Cancer Res. 2007 Sep 15;13(18 Pt 1):5341-7. doi: 10.1158/1078-0432.CCR-07-0847. Epub 2007 Sep 12.
One of the key factors that promotes angiogenesis is vascular endothelial growth factor (VEGF). Platelets are the main source of VEGF in blood and contribute to angiogenesis by release of growth factors, including VEGF, from their alpha-granules on activation. The monoclonal antibody bevacizumab blocks VEGF in the blood of patients within hours after administration. Platelets are known to endocytose plasma proteins including immunoglobulins. We tested the hypothesis that platelets take up bevacizumab.
Fluorescence-activated cell sorting analysis, immunofluorescence imaging, and Western blotting were used to study uptake and release of bevacizumab by platelets in vitro and in vivo. The angiogenic activity of platelets preincubated with bevacizumab was studied in endothelial proliferation assays. Finally, we determined whether treatment with bevacizumab neutralizes VEGF in platelets from cancer patients.
We found that platelets are able to take up bevacizumab. Activation of platelets preincubated with bevacizumab resulted in release of the antibody and release of VEGF neutralized by bevacizumab. Immunofluorescence microscopy revealed that FITC-labeled bevacizumab and P-selectin colocalize, indicating alpha-granule localization. In addition, bevacizumab uptake inhibited platelet-induced human endothelial cell proliferation. In in vivo rabbit experiments, FITC-labeled bevacizumab was present in platelets after 2 h and up to 2 weeks following i.v. administration. Finally, we found that platelets take up bevacizumab in patients receiving bevacizumab treatment. Within 8 h after bevacizumab administration, platelet VEGF was almost completely neutralized due to this uptake.
These studies show that bevacizumab is taken up by platelets and may explain its clinical effect on wound healing and tumor growth.
促进血管生成的关键因素之一是血管内皮生长因子（VEGF）。血小板是血液中VEGF的主要来源，在激活时通过从其α颗粒释放包括VEGF在内的生长因子来促进血管生成。单克隆抗体贝伐单抗在给药后数小时内可阻断患者血液中的VEGF。已知血小板可内吞包括免疫球蛋白在内的血浆蛋白。我们测试了血小板摄取贝伐单抗的假设。
采用荧光激活细胞分选分析、免疫荧光成像和蛋白质印迹法研究血小板在体外和体内对贝伐单抗的摄取和释放。在内皮细胞增殖试验中研究了与贝伐单抗预孵育的血小板的血管生成活性。最后，我们确定贝伐单抗治疗是否能中和癌症患者血小板中的VEGF。
我们发现血小板能够摄取贝伐单抗。用贝伐单抗预孵育的血小板激活后导致抗体释放以及被贝伐单抗中和的VEGF释放。免疫荧光显微镜检查显示，异硫氰酸荧光素标记的贝伐单抗与P-选择素共定位，表明其定位于α颗粒。此外，贝伐单抗摄取抑制了血小板诱导的人内皮细胞增殖。在体内兔实验中，静脉注射异硫氰酸荧光素标记的贝伐单抗后2小时至2周内，血小板中均有该标记物存在。最后，我们发现接受贝伐单抗治疗的患者血小板摄取了贝伐单抗。由于这种摄取，在贝伐单抗给药后8小时内，血小板VEGF几乎被完全中和。
这些研究表明贝伐单抗被血小板摄取，这可能解释了其对伤口愈合和肿瘤生长的临床作用。
